RNA sequences fold back on themselves to form structures that are difficult to predict, especially if only a single sequence is known (Tinoco, I.; Bustamante, C. J. Mol. Biol. 1999, 293, 271-281.; Eddy, S. R. Nature Biotechnology 2004, 22, 1457-1458; Doshi, K. J.; Cannone, J. J.; Cobaugh, C. W.; Gutell, R. R. BMC Bioinformatics 2004, 5, 105). Current algorithms correctly predict 50-70% of known base pairs on average (R. D. Dowell, S. R. Eddy, BMC Bioinformatics 5, 71 (2004) and D. H. Mathews, D. H. Turner, Curr. Opin. Struct. Biol. 16, 270 (2006). Predicted secondary structure models achieving 50-70% accuracy tend to have regions wherein the overall topology differs significantly from the correct model, making it difficult or even impossible to develop robust biological hypotheses. Knowledge of which nucleotides are likely to be paired or single-stranded can significantly improve prediction accuracies (Wilkinson et al. (2006) Nature Protocols 1:1610-1616).
Methods for visualizing the secondary structures of RNA molecules have been reported, inclusive of, for example, Chetouani et al. (1997) Nucleic Acids Res. 25:3514-3522; Hogeweg et al. (1984) Nucleic Acids Res. 12:67-74; Matzura et al. (1996) CABIOS 12:247-249; Nussinov et al. (1978) J. Appl. Math. 35:68-82; and Osterburg et al. (1981) Comput. Progr. Biomed. 13:101-109. Particularly, local nucleotide structure can be monitored using well established approaches that involve treating an RNA with chemical and enzymatic reagents (Ehresmann et al. (1987) Nucl Acids Res 15:9109-9128). These methods are widely used and can give reasonable results, especially when multiple reagents are used together or when chemical modification information is interpreted in the context of phylogenetic covariation information (Barrick et al. (2004) PNAS USA 101:6421-6426). However, current reagents used to monitor local nucleotide structure react with a subset of RNA nucleotides. Therefore, multiple reagents must be used to comprehensively analyze all four nucleotides in a given RNA. In addition, reagents currently in use exhibit widely varying nucleotide and structural selectivities such that quantitative reactivity information cannot be readily compared for the different nucleotide bases or between reagents.
In addition, denaturing slab-gel electrophoresis is an available tool for separating nucleic acids by length. However, the production and imaging of gels is a labor-intensive task, and band resolution can be poor near the origin of separation. Software that quantifies gel electrophoresis images, such as SAFA (Das et al. (2005) RNA 11:344-54) typically cannot resolve and quantify more than 200 bands per separation at single nucleotide resolution.
Therefore, there is a need in the art for methods of analyzing secondary structures of RNA molecules, by which clear and compact graphic results can be obtained quickly, accurately, and at a low cost.